UsingVibrio natriegensfor high-yield production of challenging expression targets and for protein deuteration

Abstract

ABSTRACTProduction of soluble proteins is essential for structure/function studies, however, this usually requires milligram amounts of protein, which can be difficult to obtain with traditional expression systems. Recently, the Gram-negative bacteriumVibrio natriegensappeared as a novel and alternative host platform for production of proteins in high yields. Here, we used a commercial strain derived fromV. natriegens(VmaxTMX2) to produce soluble bacterial and fungal proteins in milligram scale, which we struggled to achieve inEscherichia coli. These proteins include the cholera toxin (CT) andN-acetyl glucosamine binding protein A (GbpA) fromVibrio cholerae, the heat-labile enterotoxin (LT) fromE. coliand the fungal nematotoxin CCTX2 fromCoprinopsis cinerea. CT, GbpA and LT are secreted by the Type II secretion system in their natural hosts. When these three proteins were produced in Vmax, they were also secreted, and could be recovered from the growth media. This simplified the downstream purification procedure and resulted in considerably higher protein yields compared to production inE. coli(6– to 26-fold increase). We also tested Vmax for protein deuteration using deuterated minimal media with deuterium oxide as solvent, and achieved a 3-fold increase in yield compared to the equivalent protocol inE. coli. This is good news since isotopic labeling is expensive and often ineffective, but represents a necessary prerequisite for some structural techniques. Thus, Vmax represents a promising host for production of challenging expression targets and for protein deuteration in amounts suitable for structural biology studies.